The present invention relates to a protein containing an SRCR domain, a nucleic acid encoding such a protein and a method to produce same. In addition, this invention concerns the use of the nucleic acid and protein as well as antibodies directed against the protein.
The expression xe2x80x9cSRCRxe2x80x9d domain means xe2x80x9cscavenger receptor cysteine richxe2x80x9d domain. Such a domain comprises about 110 amino acids and is found in many proteins involved in elemental processes of the cell, e.g. cell differentiation or cell-to-cell contact. Nevertheless, these processes have not yet been understood in detail.
Therefore, it is the object of the present invention to provide a product by means of which it is possible to investigate, and optionally interfere with, elemental processes of the cells.
According to the invention this is achieved by the subject matters defined in the claims.
Therefore, the subject matter of the present invention relates to a protein containing an SRCR domain, the protein comprising the amino acid sequence of FIG. 1 (SEQ ID NO:1) or an amino acid sequence differing therefrom by one or several amino acids.
The present invention is based on the applicant""s discovery that in animals, particularly mammals, more particularly human beings, there exists a protein containing an SRCR domain, which has homologies to known proteins containing an SRCR domain but differing from these proteins on the DNA level by hybridization under conventional conditions. Such a protein comprises the amino acid sequence of FIG. 1 (SEQ ID NO:1) or an amino acid sequence differing therefrom by one or several amino acids. In addition, the applicant recognized that the protein also contains a CUB domain which also comprises about 110 amino acids. Moreover, he found that the protein can be present in tumor cells in a form other than that existing in normal cells. The modified form can present itself as additions, substitutions, inversions and/or deletions of one or several amino acids. In particular, the applicant found that the protein may have a deletion of one or several amino acids in medulloblastomas and glioblastomas and in the case of breast cancer.
The present invention refers to the above protein as xe2x80x9ca protein containing an SRCR domainxe2x80x9d (SRCR protein).
A further subject matter of the present invention relates to a nucleic acid coding for an (SRCR protein). It may be an RNA or a DNA. The latter may be e.g. a genomic DNA or a cDNA. Preferred is a DNA which comprises the following:
(a) the DNA of FIG. 1 (SEQ ID NO:1) or a DNA differing therefrom by one or several base pairs,
(b) a DNA hybridizing with the DNA from (a), or
(c) a DNA related to the DNA from (a) or (b) via the degenerated genetic code.
The expression xe2x80x9chybridizing DNAxe2x80x9d refers to a DNA which hybridizes with a DNA from (a) under normal conditions, particularly at 20xc2x0 C. below the melting point of the DNA.
The DNA of FIG. 1 (SEQ ID NO:1) was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen Gmbh, Mascheroder Weg 1b, D-38124 Braunschweig, Germany) as HFL2 under deposit accession number DSM11281 on Nov. 8, 1996 under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
A nucleic acid according to the invention is described below in the form of a DNA, particularly cDNA. It is exemplary for every nucleic acid, particularly DNA, falling under the present invention.
For the production of a cDNA according to the invention it is favorable to use mRNA from human fetal lung as a basis. Such an mRNA is known, it can be purchased e.g. from Clonetech. Full-length CDNA is generated from the mRNA, e.g. via oligodT-priming in combination with Cap-snatching. A person skilled in the art is familiar with the methods and conditions. A cDNA adapter, e.g. from the marathon kit from Clonetech, is ligated to the full-length cDNA in a blunt-end fashion. Then, the cDNA is subjected to a PCR method which uses primer pairs, one primer being specific to the cDNA adapter and the other primer being specific to DNA sequences from known proteins containing an SRCR domain. An example of the latter primer is:
cubf1 5xe2x80x2-TGCACATCTCTGAAGACCACAG-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:12)
in the 5xe2x80x2 direction, and
41nr1 5xe2x80x2-GTGGTCTGCAGGCAGCTG-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:9)
in the 3xe2x80x2 direction.
The 41nr1 primer is localized in a strongly preserved or conserved region of SRCR domains, the cubf1 primer is localized in a strongly preserved region of CUB domains. In accordance with the primer combination, an amplified cDNA is obtained which was amplified in the 5xe2x80x2 direction and 3xe2x80x2 direction, respectively. The amplified cDNA is hybridized with a labeled DNA probe specific to a nucleic acid according to the invention. Such DNA probes are e.g. the below DNA probes aime2e4f1 and a60kexf2:
DNA probe aime2e4f1: 5xe2x80x2-AGGCCAGATACTTGGCTGAC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:10)
DNA probe a60kexf2: 5xe2x80x2-CTTCAGATTACTGAAGCCCAGG-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:11)
A cDNA which hybridizes with the DNA probe aime2e4f1 and was amplified in the 5xe2x80x2 direction, is ligated with a cDNA which was amplified in the 3xe2x80x2 direction and hybridizes with the DNA probe a60kexf2. The ligation product is a cDNA according to the invention.
A cDNA according to the invention can be present in a vector and expression vector, respectively. A person skilled in the art is familiar with examples thereof. In the case of an expression vector for E. coli these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred. For the expression in yeast e.g. pY100 and Ycpad1 have to be mentioned, while e.g. pKCR, pEFBOS, cDM8 and pCEV4 have to be indicated for the expression in animal cells. The bacculovirus expression vector pAcSGHisNT-A is especially suitable for the expression in insect cells.
The person skilled in the art is familiar with suitable cells to express a cDNA according to the invention, which is present in an expression vector. Examples of such cells comprise the E. coli strains HB101, DH1, x1776, JM101, JM109, BL21 and SG13009, the latter being preferred, the yeast strain saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa as well as the insect cells sf9.
The person skilled in the art knows in which way a DNA according to the invention has to be inserted in an expression vector. He is also familiar with the fact that this DNA can be inserted in combination with a DNA coding for another protein and peptide, respectively, so that the cDNA according to the invention can be expressed in the form of a fusion protein.
In addition, the person skilled in the art knows conditions of culturing transformed cells and transfected cells, respectively. He is also familiar with processes of isolating and purifying the protein expressed by the cDNA according to the invention. Thus, such a protein, which may also be a fusion protein, is also a subject matter of the present invention.
A further subject matter of the present invention relates to an antibody directed against an above protein and fusion protein, respectively. Such an antibody can be prepared by common methods. It may be polyclonal and monoclonal, respectively. For its preparation it is favorable to immunize animalsxe2x80x94particularly rabbits or chickens for a polyclonal antibody and mice for a monoclonal antibodyxe2x80x94with an above (fusion) protein or with fragments thereof. Further xe2x80x9cboostersxe2x80x9d of the animals can be effected with the same (fusion) protein or with fragments thereof. The polyclonal antibody may then be obtained from the animal serum and egg yolk, respectively. For the preparation of the monoclonal antibody, animal spleen cells are fused with myeloma cells.
The present invention enables to investigate elemental processes of the cell. By means of a nucleic acid according to the invention, particularly a DNA, and primers derived therefrom, it can be determined in mammals, particularly human beings, whether they contain and/or express a gene which codes for an (SRCR protein). It can also be determined in which form the (SRCR protein) is present and which significance the individual forms have for the cell and processes thereof, respectively. For example, the applicant discovered that in medulloblastomas and glioblastomas as well as in breast cancer the (SRCR protein) includes a deletion. This deletion can also be identified on a genomic level. The genomic DNA clones pBa112, pBa74, pBa36, pBa41, PG141BF17, PG141BA10, pBa60, pBa101 and pBa131 are provided which in the listed order indicate a DNA range from 5xe2x80x2- greater than 3xe2x80x2 (cf. FIGS. 3, 4, and 5) which is present in normal cells but has deletions on both allels in tumor cells, particularly in cells of a medulloblastoma, glioblastoma or breast cancer. Said DNA clones also represent a subject matter of the present invention. They were also deposited with the DSMZ: pBa74 under DSM 11280, pBa36 under DSM 11272, pBa41 under DSM 11278 and pBa60 under DSM 11279, on Nov. 8, 1996 each; and PG141BF17 under DSM 11649, PG141BA10 under DSM 11648, pBa101 under DSM 11646 and pBa131 under DSM 11647, on Jul. 4, 1997 each. In addition to the coding sequence the genomic clones provide intron sequences. They are suitable for mutation analysis in tumors, e.g. as hybridization probes or with primers derived therefrom. The genomic clones thus provide diagnostic agents. For carrying out the above investigations conventional methods such as reverse transcription, PCR reaction, hybridization and sequencing can be used. According to the invention a kit is also provided which contains an above nucleic acid, particularly DNA, and/or primers derived therefrom as well as carriers and conventional auxiliary agents. Moreover, diagnostic steps can be taken with an (SRCR protein) according to the invention. In particular, it can be determined whether autoantibodies exist against such a protein.
Furthermore, the present invention is suitable to interfere with elemental processes of the cell. An (SRCR protein) can be inserted in mammals, particularly human beings. For this purpose, it may be favorable to couple the (SRCR protein) to a protein which is not considered foreign by the respective body, e.g. transferrin or BSA. A nucleic acid according to the invention, particularly a DNA, can also be inserted and expressed in mammals, particularly human beings. For this purpose, it may be favorable to control the expression of the nucleic acid according to the invention by a tissue-specific promoter. The expression of an (SRCR protein) can be controlled and regulated by an antibody according to the invention.
Thus, the present invention represents a great contribution to the diagnostic and therapeutic detection of elemental processes of the cell. In particular, tumoral diseases, particularly of the central nervous system, e.g. medulloblastomas or glioblastomas, as well as the breast can be diagnosed on a genetic level and steps can be taken thereagainst. The diagnostic detection of elemental processes of the cell cannot only be made postnatally but also already prenatally in this connection.